Expression In Pancreatic Tissues Research Articles

Efficient Gene Delivery and Expression in Pancreas and Pancreatic Tumors by Capsid-Optimized AAV8 Vectors.

Despite efforts to use adeno-associated viral (AAV) vector-mediated gene therapy for treatment of pancreatic ductal adenocarcinoma (PDAC), transduction efficiency remains a limiting factor and thus improvement of AAV delivery would significantly facilitate the treatment of this malignancy. Site-directed mutagenesis of specific tyrosine (Y) residues to phenylalanine (F) on the surface of various AAV serotype capsids has been reported as a method for enhancing gene transfer efficiencies. In the present studies, we determine whether Y-to-F mutations could also enhance AAV8 gene transfer in the pancreas to facilitate gene therapy for PDAC. Three different Y-to-F mutant vectors (a single-mutant, Y733F; a double-mutant, Y447F+Y733F; and a triple-mutant, Y275F+Y447F+Y733F) and wild-type AAV8 (WT-AAV8) were administered by intraperitoneal or tail-vein routes to KrasG12D+/-, KrasG12D+/-/Pten+/-, and wild-type mice. The transduction efficiency of these vectors expressing the mCherry reporter gene was evaluated 2 weeks post administration in pancreas or PDAC and correlated with viral genome copy numbers. Our comparative and quantitative analyses of the transduction profiles demonstrated that the Y-to-F double-mutant exhibited the highest mCherry expression in pancreatic tissues (range 45-70%) compared with WT-AAV8 (7%; p < 0.01). We also detected a 7-fold higher level of vector genome copy numbers in normal pancreas following transduction with the double-mutant AAV8 compared with WT-AAV8 (10,285 vs. 1,500 vector copies/μg DNA respectively, p < 0.05). In addition, we observed that intraperitoneal injection of the double-mutant AAV8 led to a 15-fold enhanced transduction efficiency as compared to WT-AAV8 in mouse PDAC, with a corresponding ∼14-fold increase in vector genome copy numbers (26,575 vs. 2,165 copies/μg DNA respectively, p < 0.05). These findings indicate that the Y447+Y733F-AAV8 leads to a significant enhancement of transduction efficiency in both normal and malignant pancreatic tissues, suggesting the potential use of this vector in targeting pancreatic diseases in general, and PDAC in particular.

The Effect of Three Months of Resistance Training on TCF7L2 Expression in Pancreas Tissues of Type 2 Diabetic Rats

Background: Resistance exercise is recommended as a useful therapeutic tool for the treatment of type 2 diabetes (T2D); however, the frequency of studies is inadequate to establish the precise mechanisms of any association between them. Objectives: In this study, we aimed to assess the effect of three months of resistance training on TCF7L2 expression in pancreatic tissues, serum insulin and glucose. Materials and Methods: For this purpose, type 2 diabetes (T2D) was induced by intraperitoneal streptozotocin-nicotinamide in eighteen male Wistar rats aged 10 weeks (22030 g). Then, the rats were randomly divided into exercise and control groups. The exercise rats completed a three-month resistance training intervention that included climbingona stepladder for 5 days weekly. The control group did not participate in exercise intervention. Fasting glucose and insulin were measured before and after injection (7 days) and after intervention. TCF7L2 gene expression of pancreatic tissues was measured in both groups after the exercise treatment, and the ratio between the two groups was calculated. Results: Fasting glucose increased and serum insulin decreased significantly by T2D induction in the two groups at baseline. Resistance training resulted in a decrease in fasting glucose and an increase in insulin in exercise rats. Data also showed that TCF7L2 gene expression decreased after resistance training compared with the control group. Conclusions: Based on these data, increased serum insulin can be attributed to a decrease in TCF7L2 gene expression of pancreatic cells by resistance training in T2D rats.

Effect of tiron on remote organ injury in rats with severe acute pancreatitis induced by L-arginine.

Acute pancreatitis (AP) is an acute inflammatory disorder of the pancreas that can be complicated by involvement of other remote organs. Oxidative stress is known to have a crucial role in the development of pancreatic acinar damage and one of the main causes in multisystem organ failure in experimental AP. The aim of the study was to determine the effect of tiron on pancreas and remote organ damage in L-arginine (L-Arg) induced AP rat model. Thirty-two male rats were divided in random into four groups: control, tiron, L-Arg, and tiron with L-Arg. At the end of the experiment, blood samples were withdrawn for biochemical analysis. The pancreas, lung, kidney, and liver were collected for histopathological examination. Estimation of pancreatic water content was done. Analysis of pulmonary, hepatic, renal, and pancreatic lipid peroxide levels (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) were carried out. Finally, nuclear factor kappa B (NF-κB) and transforming growth factor β1 (TGF-β1) expression in pancreatic tissue was determined. Results indicated that treatment with tiron significantly decreased lipid peroxide levels and markedly increased both SOD activity and GSH level. Moreover, histopathological analysis further confirmed that administration of tiron relatively ameliorates pancreatic acinar cells and remote organ damage. Increased immunoreactivity of NF-κB and TGF-β1 were reduced also by tiron treatment. These findings pointed out the protective role of the mitochondrial antioxidant, tiron against AP induced by L-Arg.

Salivary HOTAIR and PVT1 as novel biomarkers for early pancreatic cancer.

Sensitive and non-invasive biomarkers for pancreatic cancer (PC) are lacking. We aimed to identify salivary long non-coding RNAs (lncRNAs) as biomarkers in diagnosis of resectable PC. Five well-documented lncRNAs: H19, HOTAIR, HOTTIP, MALAT1, PVT1, which are most closely associated with pancreatic cancer from previous studies were selected as putative lncRNA biomarkers. Their expression in pancreatic tissues and saliva of cancer patients and healthy controls was measured by quantification polymerase chain reaction (qPCR). Compared with benign pancreatic tumour (BPT) and normal pancreatic tissues (NPT), HOTAIR, HOTTIP and PVT1 were significantly up-regulated in pancreatic cancer tissues (PCT). As compared to BPT or healthy groups, the salivary levels of HOTAIR and PVT1 were significantly higher in PC group. They were significantly reduced after the curative pancreatectomy. Both salivary lncRNAs distinguished PC patients from healthy controls and BPT patients with sensitivities and specificities ranging from 60–97%. The expression of salivary HOTAIR and PVT1 did not differ significantly between healthy controls and any one of eight leading cancers worldwide. Collectively, our findings indicate that salivary HOTAIR and PVT1 show potential as novel non-invasive biomarkers for detecting PC.

Mallotus roxburghianus modulates antioxidant responses in pancreas of diabetic rats

Mallotus roxburghianus has long been used by Mizo tribal people for the treatment of diabetes. Scientific validation at known doses may provide information about its safety and efficacy. Methanolic leaf extract of M. roxburghianus (MRME 100 and 400mg/kg) was tested in comparison with normal and alloxan diabetic rats for 28 days p.o. in terms of body and pancreatic weight, blood glucose level, antioxidant enzymes, expression of visfatin and PCNA, histopathology and histomorphometric measurements of pancreas. The results were evaluated statistically using ANOVA, correlation and regression and Principal component analysis (PCO). MRME (100 and 400mg/kg) treatment significantly (p<0.0001) decreased the body weight, blood glucose level, improved the mass and size of pancreas, elevated the levels of antioxidant enzymes and up regulate the expression of visfatin and PCNA. PCO analysis was good to fitness and prediction distinguishes the therapeutic effects of M. roxburghianus from the alloxan induced diabetic rats. MRME has significant role in protecting animals from alloxan-induced diabetic oxidative stress in pancreas and exhibited promising antihyperglycaemic and antioxidant activities along with significant reversal of disturbed antioxidant status and lipid peroxidative damage. Pancreatic architecture and physiology under diabetic oxidative stress have been significantly modulated by MRME and validated as a drug candidate for antidiabetic treatment. M. roxburghianus treatment restores the antioxidant enzyme system and rejuvenates the islets mass in alloxanized rat by accelerating visfatin and PCNA expression in pancreatic tissue.

Pterostilbene as treatment for severe acute pancreatitis.

Acute pancreatitis (AP) has a fast onset and progression, which lead to an unfavorable prognosis. Therefore, the development of novel drugs for its treatment is critical. As a homologous derivative of resveratrol, pterostilbene exerts a variety of effects including anti-inflammatory, antioxidant, and antitumor effects. This study investigated the potential of pterostilbene for treatment of severe AP (SAP) and related mechanisms. Effects of pterostilbene were evaluated in a Wistar rat model of AP. Serum levels of amylase (AMY), creatinine (Cr), and alanine aminotransferase (ALT) were quantified. Furthermore, serum levels of tumor necrosis factor (TNF)-a and interleukin (IL)-1b were quantified using enzyme-linked immunosorbent assay. Nuclear factor (NF)-kB expression in pancreatic tissues was quantified by real-time PCR and western blotting. The production of reactive oxygen species (ROS) was determined using a spectrometer, while superoxide dismutase (SOD) activity was assayed. In the AP rat model, the expression of inflammatory markers TNF-a and IL-1b, expression of NF-kB, and serum indices (AMY, Cr, and ALT) increased compared to the corresponding levels in the control group (P < 0.05). Pterostilbene reduced serum levels of TNF-a and IL-1b; decreased NF-kB gene expression, serum indices, and ROS generation; and increased SOD activity in a dose-dependent manner. In conclusion, pterostilbene can alleviate SAP-induced tissue damage by decreasing the inflammatory response and by promoting antioxidation leading to the protection of pancreatic tissues.

Effect of gentiopicroside on experimental acute pancreatitis induced by retrograde injection of sodium taurocholate into the biliopancreatic duct in rats

Gentiopicroside (otherwise known as Gentiopicrin), one of the main active ingredients from the traditional Chinese herb medicine Gentiana manshurica Kitag, presents the effect of attenuating acute pancreatitis in rats. The experimental acute pancreatitis was made by retrograde injection of sodium taurocholate into the biliopancreatic duct in rats. Gentiopicroside was given orally and it markedly reduced the pancreatitis-evoked increase of serum amylase and lipase activity, decreased the pancreas mass/body mass index, tissue water content, TNF-α and IL-1β concentrations, and attenuated the histopathological changes and NF-κB p65 protein expression in pancreatic tissue. The results indicate that the function of gentiopicroside on acute pancreatitis may be related to inhibiting the release of inflammatory mediators and NF-κB p65 protein expression.

Vascular Endothelial Growth Factor, a Novel and Highly Accurate Pancreatic Fluid Biomarker for Serous Pancreatic Cysts

Mucinous pancreatic cysts (intraductal papillary mucinous neoplasm and mucinous cystic neoplasm) have the potential to progress to invasive pancreatic adenocarcinoma, presenting an opportunity for early detection, prevention, and cure. Serous cystic neoplasms (SCN) have no malignant potential, but can mimic mucinous pancreatic cysts on imaging. Therefore, identification of biomarkers that can distinguish between cystic lesions is critically important. We hypothesize that vascular endothelial growth factor (VEGF)-A levels in pancreatic fluid correlate with pathologic diagnosis. Pancreatic cyst/duct fluid samples were prospectively collected from patients undergoing pancreatic resection and correlated with surgical pathology. VEGF levels were detected by ELISA. VEGF-A and VEGF receptor 2 expression in pancreatic tissue was localized by immunohistochemistry. Genetic alterations of the von Hippel-Lindau gene were determined by targeted next-generation sequencing. Eighty-seven patients met inclusion criteria for enrollment. Final pathologic diagnoses included pseudocyst (n = 9), SCN (n = 17), mucinous cystic neoplasm (n = 24), low/moderate grade intraductal papillary mucinous neoplasm (n = 16), high-grade/invasive intraductal papillary mucinous neoplasm (n = 10), and pancreatic ductal adenocarcinoma (n = 11). VEGF-A was significantly upregulated in SCN cyst fluid compared with all other diagnoses (p < 0.0001). With a cut-off of 8,500 pg/mL, VEGF-A has 100% sensitivity and 97% specificity as an SCN biomarker. VEGF-A and VEGF receptor 2 are overexpressed in SCN cyst tissue. VEGF-C was also significantly elevated in SCN cyst fluid (p < 0.0001). With a cut-off set at 200 pg/mL, VEGF-C identifies SCN with 100% sensitivity and 90% specificity. The presence of a von Hippel-Lindau mutation in SCN cyst tissue correlates with elevated cyst fluid VEGF levels. This is the first report of a cyst fluid protein biomarker that can positively identify SCN. The ability to distinguish SCN from premalignant/malignant pancreatic cysts can spare the cost and risk of surveillance and surgical intervention in select patients.

Effects of Jiang Tang Fang Long Formula on Insulin Production and Function in an Animal Model of Diabetic Hearing Loss

ObjectiveTo study the influence of Jiang Tang Fang Long formula on insulin production and function in an animal model of diabetic hearing loss. MethodsWistar rats (n=60) were randomly divided into 6 groups (10 in each) to receive no treatment (the normal control, Group A), or to receive intra-peritoneal 55 mg/kg streptozotocin with (Groups C, D and E) or without (Group B) subsequent Jiang Tang Fang Long formula treatment at various doses or Yu Long Wan treatment (Group F). After 60 days, fasting blood glucose (FBG), body weight (BW) and fasting insulin (FINS) were recorded and the HOMA-IR and HOMA-β calculated. Insulin expression in pancreatic tissues was measured by radioimmunoassay. ResultsCompared with animals that received streptozotocin without rescue treatment (Group B), animals that received higher doses of Jiang Tang Fang Long formula(Groups D and E) showed improved indices of diabetes manifestation (P< 0.05) and improved HOMA-β (P<0.05) in a dose-dependent manner, as well as improved insulin expression in pancreatic islets (P<0.05). The difference between low dose Jiang Tang Fang Long formula treatment (Group C) and Group B was not significant (P>0.05). ConclusionOur results suggest that Jiang Tang Fang Long formula may improve pancreatic β-cells function which may explain its efficacy in treating diabetic hearing loss.

Galectin-3 Deficiency Accelerates High-Fat Diet–Induced Obesity and Amplifies Inflammation in Adipose Tissue and Pancreatic Islets

Obesity-induced diabetes is associated with low-grade inflammation in adipose tissue and macrophage infiltration of islets. We show that ablation of galectin-3 (Gal-3), a galactoside-binding lectin, accelerates high-fat diet–induced obesity and diabetes. Obese LGALS3−/− mice have increased body weight, amount of total visceral adipose tissue (VAT), fasting blood glucose and insulin levels, homeostasis model assessment of insulin resistance, and markers of systemic inflammation compared with diet-matched wild-type (WT) animals. VAT of obese LGALS3−/− mice exhibited increased incidence of type 1 T and NKT lymphocytes and proinflammatory CD11c+CD11b+ macrophages and decreased CD4+CD25+FoxP3+ regulatory T cells and M2 macrophages. Pronounced mononuclear cell infiltrate, increased expression of NLRP3 inflammasome and interleukin-1β (IL-1β) in macrophages, and increased accumulation of advanced glycation end products (AGEs) and receptor for AGE (RAGE) expression were present in pancreatic islets of obese LGALS3−/− animals accompanied with elevated phosphorylated nuclear factor-κB (NF-κB) p65 and mature caspase-1 protein expression in pancreatic tissue and VAT. In vitro stimulation of LGALS3−/− peritoneal macrophages with lipopolysaccharide (LPS) and saturated fatty acid palmitate caused increased caspase-1–dependent IL-1β production and increased phosphorylation of NF-κB p65 compared with WT cells. Transfection of LGALS3−/− macrophages with NLRP3 small interfering RNA attenuated IL-1β production in response to palmitate and LPS plus palmitate. Obtained results suggest important protective roles for Gal-3 in obesity-induced inflammation and diabetes.

Differential gene expression in pancreatic tissues of streptozocin-induced diabetic rats and genetically-diabetic mice in response to hypoglycemic dipeptide cyclo (His-Pro) treatment

Diabetic studies are mostly interested in gene expression in the pancreas, the site of insulin secretion that regulates blood glucose levels. However, a single gene approach has been ruled out for many years in discovering new genes or the molecular networks involved in the induction process of diabetes. To understand the molecular mechanisms by which cyclo (His-Pro) (CHP) affects amelioration of diabetes mellitus, we performed gene expression profiling in the pancreatic tissues of two diabetic animal models, streptozocin (STZ)-induced diabetic rats (T1DM) and genetically-diabetic (C57BL/6J ob/ob) mice (T2DM). To understand the healing process of these diabetic rodents, we examined the effects of CHP on various gene expression in pancreatic tissues of both animal models. Our microarray analysis revealed that a total of 1,175 genes were down-regulated and 629 genes were up-regulated in response to STZ treatment, and the altered expression levels of numerous genes were restored to normal state upon CHP treatment. In particular, 476 genes showed significantly altered gene expression upon CHP treatment. In a functional classification, 7,198 genes were counted as differentially expressed in pancreatic tissues of STZ- and CHP-treated rats compared with control, whereas 1,534 genes were restored to normal states by CHP treatment. Microarray data demonstrated for the first time that overexpression of the genes encoding IL-1 receptor, lipid metabolic enzymes (e.g. Mte1, Ptdss1, and Sult2a1), myo-inositol oxygenase, glucagon, and somatostatin as well as down-regulation of olfactory receptor 984 and mitochondrial ribosomal protein, which are highly linked to T1DM etiology. In genetically-diabetic mice, 4,384 genes were altered in gene expression by more than 2-fold compared to the control mice, when counted differentially expressed. In genetically-diabetic mice, 4,384 genes altered in expression by higher than 2-fold were counted as differentially expressed genes in pancreatic tissues of CHP-treated mice. On the other hand, 2,140 genes were up-regulated and 2,244 genes were down-regulated by CHP treatment. The results of the microarray analysis revealed that up-regulation of IL-2, IL12a, and leptin receptor and down-regulation of PIK3 played important physiological roles in the onset of T2DM. In conclusion, we hypothesize that CHP accelerates alterations of gene expression in ameliorating diabetes and antagonizes those that induces the disease.

Hypoglycemic and anti apoptotic effect of garlic in streptozotocin induced diabetic rats

Diabetes mellitus is caused by many factor include oxidative stress that leads to apoptosis of beta cells of the pancreas and so the antioxidant therapy strongly correlated with decrease risk of diabetes mellitus. The aim of the present study was to investigate the efficacy of an aqueous extract of raw garlic in controlling serum glucose, plasma c peptide of insulin, level of reduced glutathione and catalase activity in pancreatic tissue, also to estimate caspase 3 activity expression in pancreatic tissue in streptozotocin induced diabetic rats treated daily with garlic extract intraperitoneally (IP) for 6 weeks. This study was carried on 30 rats: grouped into 3 group. Group 1, the control normal group, was injected IP daily with 0.5 ml saline and group 2; diabetic group was injected with streptozotocin, 60 mg/Kg body weight (BWt) IP in 0.5 ml saline once and group 3; garlictreated group, was injected IP daily with 500 mg/kg of the garlic extract 2 weeks before streptozotocin and 4 week after streptozotocin injection. There was a significant increase in blood glucose in streptozotocin group II (p = 0.001) as compared with control groups (331.3 &plusmn; 16.15 vs 101.8 &plusmn;4.02 mg/dl) respectively and significantly decreased after treatment with garlic extract (161.5 &plusmn; 5.28 mg/dl). C peptide was significantly decreased in streptozotocin group II (p = 0.001) as compared with control groups (0.034 &plusmn; 0.003 vs 0.053&plusmn;0.001 ng/ml) respectively and significantly increased after treatment with garlic extract (0.046 &plusmn; 0.003). Catalase activity of pancreatic tissue was significantly decreased in streptozotocin group (p = 0.001) as compared with control groups (11.10 &plusmn; 0.73 vs 25.7 &plusmn; 0.55 U/gm tissue) respectively and significantly increased after treatment with garlic extract (20.3 &plusmn; 0.66). Reduced glutathione content of pancreatic tissue was significantly decreased in streptozotocin group (p = 0.001) as compared with control groups (0.67 &plusmn; 0.055 vs 1.23 &plusmn; 0.076 mg/g tissue) respectively and significantly increased after treatment with garlic extract (0.89 &plusmn; 0.080 mg/g tissue). Also it was observed that the expression of caspase 3 protein in the pancreatic tissue was decreased after garlic treatment using western blot technique. These results revealed that aqueous extract of raw garlic may have antioxidant and antiapoptotic activity that could be used in treatment of diabetes mellitus.Key word: Diabetes mellitus, STZ, antioxidant, Caspase, garlic

Abstract 4315: Transgenic expression of human Thymidylate Synthase cooperates with an Ink4a/Arf mutation to enhance tumor progression

Abstract While thymidylate synthase (TS) is an established tumor prognostic biomarker and therapeutic target, it was only recently shown to exhibit an unexpected direct role in tumorgenesis. Overexpression of catalytically active human TS (hTS) transformed rodent cells in vitro and in vivo, while TS missence mutants with loss of enzymatic function were non-transforming (Cancer Cell v5, p341, 2004). In addition, transgenic hTS mice developed hyperplasia and tumors of the endocrine pancreas (Oncogene v26, p4817, 2007). These mice showed high levels of hTS expression in pancreatic tissue and lower levels in most other organs. Since hTS transgenic animals developed islet cell adenomas after long latency, we asked whether deregulated hTS would cooperate with other genetic lesions in the initiation and progression of tumor development. To address this, we have crossed hTS transgenic animals with Ink4a/Arf null mice and evaluated tumor spectra, incidence, latency and tumor burden. The Ink4a/Arf null control mice developed predominantly lymphomas and fibrosarcomas while the hTS/Ink4a/Arf -/- mice develop liposarcoma, rabdomyosarcoma, teratoma, hystiocytic neoplasms as well as fibrosarcoma and lymphoma. Soft tissue sarcomas (excluding fibrosarcoma) were large and developed only in hTS/Ink4a/Arf -/- mice ranging from 2.8 to 10 grams. In addition, tumors isolated from hTS/Ink4a/Arf -/- mice contained blood filled cyst-like structures in the lymph nodes, liver, lung, spleen and pancreas as well as on the surface of teratoma and liposarcoma suggesting increased or dysplastic angiogenesis. The frequency of histiocytic neoplasms in hTS/Ink4a/Arf -/- mice was significantly enhanced (42%) as compared to control mice (8%) and these tumors spread to multiple sites (as many as 9 different organs) as compared to the control mice where tumors were localized predominantly in lymph nodes and spleen. Similarly, lymphomas spread to many extranodal organs, while the control tumors were localized predominantly to lymph nodes or spleen. In summary, we observed that transgenic expression of hTS in Ink4a/Arf null mice accelerated tumor growth leading to more aggressive disease with higher tumor burden. Understanding the mechanism of TS-mediated tumor progression may lead to development of new agents that may act synergistically with known TS inhibitors. The observation that TS promotes tumor growth and it is not just a passive biomarker for cell proliferation will have an important impact for the prevention and treatment of human cancers given the widespread over-expression of TS in the common adult tumors including lymphomas, colorectal, breast, pancreatic, sarcomas and lung cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4315. doi:10.1158/1538-7445.AM2011-4315

Influence of baicalin on TNF-α mRNA, caspase-3 and P-selectin expression in pancreatic tissue of rats with severe acute pancreatitis

Baicalin reduces the severity of severe acute pancreatitis (SAP) in a rat model. This study was carried out to examine the effect of baicalin on TNF-alpha mRNA, caspase-3 and P-selectin protein expression in the pancreas of rats with SAP. Rats with SAP were randomly assigned to untreated, baicalin-treated, octreotide-treated and sham-operated group. The TNF-alpha mRNA expression level, caspase-3 and P-selectin protein expression levels of pancreatic tissue were measured 3, 6 and 12 hours after operation. The caspase-3 protein expression level was significantly higher in the treated groups than that in the untreated group at 3 hours (p <0.01). P-selectin protein expression level in pancreas of the treated groups was significantly lower than that of the untreated group (p <0.05 or <0.01). TNF-alpha mRNA expression level in pancreas decreased in baicalintreated group while untreated group increased at different time points (p <0.05). Baicalin increases caspase-3 expression and inhibits TNF-alpha mRNA and P-selectin protein expression in pancreatic tissue of SAP rats.

Impact of Pancreatic Cold Preservation on Rat Islet Recovery and Function

Islet transplantation success depends on the number and quality of islets transplanted. This study aimed at exploring the molecular mechanisms associated with cold pancreas preservation and their impact on islet cell survival and function. Rat pancreata were stored in cold University of Wisconsin preservation solution for short (3 hr; control) or long (18 hr) cold ischemia times (CIT). Pancreata exposed to long CIT yielded lower islet numbers and showed reduced cellular viability; isolated islets displayed higher levels of phosphorylated stress-activated protein kinase (c-jun N-terminal Kinase and Mitogen-Activated Protein Kinase-p38), and chemokine (C-C) ligand-3, and lower levels of vascular endothelial growth factor, interleukins (IL)-9 and IL-10. Islets obtained from long-CIT pancreata were functionally impaired after transplantation. Differential proteomic expression in pancreatic tissue after CIT included increased eukaryotic translation elongation factor-1-alpha-1 (apoptosis related) and reduced Clade-B (serine protease inhibitor). Our study indicates that cold ischemia stimulates inflammatory pathways (chemokine (c-c)ligand-3, phosphorylation of c-jun N-terminal Kinase and mitogen-activated protein kinase-p38, and eukaryotic translation elongation factor-1-alpha-1) and decreases repair/cytoprotective pathways (IL-10, vascular endothelial growth factor, and Clade-B), all of which may negatively affect the quality and mass of islets obtained from a donor pancreas.

The Influence of Brain Death on Tissue Factor Expression in the Pancreatic Tissues and Isolated Islets in Rats

Introduction Tissue factor (TF) in islets has been identified as the main trigger of the instant blood-mediated inflammatory reaction. Because the crucial events that directly induce TF remain to be determined, we focused on the influence of brain death (BD) on TF expression in pancreatic tissues and isolated islets. Materials and Methods BD was induced in male Lewis rats weighing 250–300 g by inflation of a Fogarty catheter placed intracranially. The rats were mechanically ventilated for 6 hours until removal of the pancreas. The expression of TF protein in pancreatic tissues was examined using Western blotting assay. Messenger RNA (mRNA) expressions of TF in pancreatic tissue and isolated islets were analyzed using real-time polymerase chain reaction (PCR) assay. The influence of BD on the isolation outcome was evaluated by islet yield, purity, viability, and function. Results TF protein and mRNA levels in the pancreatic tissues were similar between the groups. However, TF mRNA in the isolated islets of the BD group was significantly greater than that of the control group ( P = .04). Islet yield was considerably lower, and purity significantly lower in the BD than the control group ( P = .002). Unexpectedly, ATP/DNA ratio and respiratory activity were comparable between the groups. Conclusions Although BD per se was not sufficient to induce TF expression in pancreatic tissues, BD combined with subsequent warm ischemic damage during isolation procedures remarkably up-regulated TF expression in isolated islets, suggesting that BD is of great importance as an initiator of TF induction in the islet grafts. The present study demonstrated that the expression of inflammatory mediators rather than islet viability is more susceptible to BD.

Alteration of Cpn60 expression in pancreatic tissue of rats with acute pancreatitis

The expression of heat-shock protein 60 (also known as chaperonin 60, Cpn60) in experimental acute pancreatitis (AP) is considered to play an active role in the prevention of abnormal enzyme accumulation and activation in pancreatic acinar cells. However, there are controversial results in the literature regarding the relationship between the abnormality of Cpn60 expression and AP onset and development. The purpose of this study was to investigate the alternations of Cpn60 expression and the relationship between the abnormal expression of Cpn60 and AP progression in rat severe acute pancreatitis (SAP) models. In this report, we induced SAP in Sprague-Dawley (SD) rats by reverse injection of sodium deoxycholate into the pancreatic duct, and examined the dynamic changes of Cpn60 expression in pancreatic tissues from different time points and at different levels with techniques of real-time PCR, western blotting, and immunohistochemistry. At 1 h after SAP induction, the expression of Cpn60 mRNA in the AP pancreatic tissues was higher than those in the sham-operation group and normal control group, but decreased sharply as the time period was extended, and there was a significant difference between 1 h and 10 h after SAP induction (p < 0.05). In the AP process, Cpn60 protein expression showed transient elevation as well, and the increased protein expression occurred predominantly in affected, but not totally destroyed, pancreatic acinar cells. As AP progressed, the pancreatic tissues were seriously damaged, leading to a decreased overall Cpn60 protein expression. Our results show a complex pattern of Cpn60 expression in pancreatic tissues of SAP rats, and the causality between the damage of pancreatic tissues and the decrease of Cpn60 level needs to be investigated further.

S100A6 Is Increased in a Stepwise Manner during Pancreatic Carcinogenesis: Clinical Value of Expression Analysis in 98 Pancreatic Juice Samples

There are few reports describing the diagnostic significance of S100A6 expression in clinical samples obtained from patients with pancreatic disease. In the present study, we measured S100A6 expression in pancreatic tissues and juice to evaluate its involvement in pancreatic carcinogenesis. We did quantitative real-time reverse transcription-PCR to measure mRNA expression in microdissected cells and pancreatic juice samples. Microdissected invasive ductal carcinoma and intraductal papillary mucinous neoplasm (IPMN) cells expressed significantly higher levels of S100A6 than did microdissected pancreatitis-affected epithelial and normal cells (all comparison; P < 0.008). Median levels of S100A6 in invasive ductal carcinoma were higher than those in IPMN, and those in pancreatitis-affected epithelial cells tended to be higher than those in normal cells, although these differences were not statistically significant. In analyses of pancreatic juice, IPMN and pancreatic cancer samples expressed significantly higher levels of S100A6 than did chronic pancreatitis samples (both; P < 0.017), but levels in pancreatic cancer and IPMN samples did not differ form each other. Receiver operating characteristic (ROC) curve analysis revealed that measurement of S100A6 was useful for discriminating cancer (area under the ROC curve, 0.864) or IPMN (area under the ROC curve, 0.749) from chronic pancreatitis. The present data suggest that expression of S100A6 is increased in a stepwise manner during pancreatic carcinogenesis and may be a biomarker for evaluating malignant potential. Measurement of S100A6 in pancreatic juice may be useful to detect early pancreatic cancer or identify individuals with high-risk lesions that may progress to pancreatic cancer.

Significance of platelet activating factor receptor expression in pancreatic tissues of rats with severe acute pancreatitis and effects of BN52021

To investigate the dynamic changes and significance of platelet activating factor receptor (PAF-R) mRNA and protein in pancreatic tissues of rats with severe acute pancreatitis (SAP) and effects of BN52021 (Ginkgolide B). Wistar male rats were randomly assigned to the negative control group (NC group), SAP model group (SAP group), and BN52051-remedy group (BN group), and each of the groups was divided into 6 subgroups at different time points after operation (1 h, 2 h, 3 h, 6 h, 12 h, and 24 h) (n = 10 in each). PT-PCR and Western blot methods were used to detect PAF-RmRNA and protein expression in pancreatic tissues of rats respectively. Pathological examination of pancreatic tissues was performed and the serum amylase change was detected. Serum amylase and pathological results showed the that SAP model was successfully prepared, BN52021 was able to decrease serum amylase, and the pathological ratings in BN group at 3 h, 6 h, and 12 h significantly decreased compared with those in the SAP group (8.85 +/- 0.39 vs 5.95 +/- 0.19, 9.15 +/- 0.55 vs 5.55 +/- 0.36, 10.10 +/- 0.65 vs 6.72 +/- 0.30, P < 0.05). The result of PAF-mRNA showed dynamic changes in SAP and BN groups, which increased gradually in early stage, reached a peak at 3 h (0.71 +/- 0.14 vs 0.54 +/- 0.14, 0.69 +/- 0.13 vs 0.59 +/- 0.04, P < 0.05), and decreased gradually later. There were significant differences at each time point except 1 h and 2 h, when compared with those in the NC group (0.71 +/- 0.14 or 0.69 +/- 0.13 vs 0.47 +/- 0.10, 0.38 +/- 0.08 or 0.59 +/- 0.04 vs 0.47 +/- 0.09, 0.25 +/- 0.07 or 0.29 +/- 0.05 vs 0.46 +/- 0.10, 0.20 +/- 0.06 or 0.20 +/- 0.04 vs 0.43 +/- 0.09, P < 0.05), whereas there was no significant difference between BN and SAP groups at each time point. The result of PAF-R protein showed that the change of PAF-R protein in the SAP group and the BN group was consistent with that of PAF-R mRNA. There were significant differences at each time point except 1 h, when compared with those in the NC group (0.90 +/- 0.02 or 0.80 +/- 0.05 vs 0.48 +/- 0.02, 1.69 +/- 0.06 or 1.58 +/- 0.02 vs 0.48 +/- 0.03, 1.12 +/-0.10 or 0.98 +/- 0.03 vs 0.49 +/- 0.09, 1.04 +/- 0.14 or 0.87 +/- 0.02 vs 0.52 +/- 0.08, 0.97 +/- 0.16 or 0.90 +/- 0.05 vs 0.49 +/- 0.10, P < 0.05), whereas there was no significant difference between the BN group and the SAP group. PAF-R plays an important role in occurrence and development of SAP. BN52021 exerts biological effects through competitively inhibiting the binding of increased both PAF and PAF-R expression rather than through decreasing PAF-R expression in pancreatic tissues.

Effect of BN52021 on NFκ-Bp65 expression in pancreatic tissues of rats with severe acute pancreatitis

To investigate dynamic changes and significance of expression of NF-kappaBp65 in pancreatic tissues of rats with severe acute pancreatitis (SAP), as well as BN52021 effects. Wistar male rats were randomly divided into negative control group (NC group, n=60), SAP-model group (SAP group, n=60), and BN52021-treated group (BN group, n=60), and each of the above groups was respectively divided into 6 subgroups at different time points after operation (1 h, 2 h, 3 h, 6 h, 12 h, and 24 h) (n=10). By RT-PCR and Western blot, NF-kappaBp65 mRNA and its protein expression in pancreatic tissues of rats were detected respectively. The expression of NF-kappaBp65 mRNA dynamically changed in both SAP groups and BN groups. The mRNA level was higher in SAP groups than NC groups at 2 h, 3 h, 12 h, and 24 h after operation (P<0.05), higher in BN groups than NC groups at all time points (P<0.05), and higher in BN groups than SAP group at 1 h (P<0.05). The NF-kappaBp65 protein level was higher in SAP groups than NC groups at 1 h, 3 h, and 6 h (P<0.01), and 2 h, 12 h, and 24 h (P<0.05), higher in BN groups than NC groups at all time points (P<0.05), and lower in BN groups than SAP groups at 1 h, 3 h, and 6 h (P<0.05). The expression of NF-kappaBp65 in pancreatic tissues is dynamically changed and the changes play an important role in pathogenesis of SAP. BN52021 exerts therapeutic effects through reducing the expression level of NF-kappaBp65 protein in the early stage of SAP.